Unlike simple retroviruses, human and simian immunodeficiency viruses (HIV/SIV) encode several accessory proteins which are packaged into the virus particle (Vpr, Vpx, Vif). In the last budget period, their role in HIV/SIV replication has been studied with a focus on their function as virion components. Several findings were made including the discovery that nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes (i.e., Vpx and Vpr) in HIV-2/SIVSM; the discovery that the HIV-2/SIVSM Vpx gene is of SIVAGM origin and was acquired by non-homologous recombination; the finding that Vif is a component of the HIV/SIV core; the finding that virion associated accessory proteins can be used to target foreign proteins to the virus particle; the discovery that integrase (IN) mutant HIV-1 proviruses can be functionally complemented by packaging wildtype integrase as a Vpr fusion protein; and the demonstration that certain Vpr/x-based fusion proteins exert a significant antiviral effect when packaged into virions. In this renewal application, the investigators propose to continue to study virion associated accessory proteins, focusing in particular on their ability to target heterologous proteins into the HIV/SIV virion. Two main goals will be pursued including the identification and characterization of fusion partners which disrupt the function of other virion components and to use Vpr/Vpx/Vif based fusion proteins to specifically probe functional relationships of viral and/or cellular proteins within the virus particle. Three specific aims are proposed including to characterization of bacterial staphylococcal nuclease (SN) as a candidate antiviral fusion partner; to develop further Vpr and Vpx based fusion proteins for which an antiviral activity has already been demonstrated and to examine additional fusion partners for their activity within virus particles. Using this unique approach, the investigators expect to gain new insights into virion architecture and the structure/function relationships of virion components.